Discrimination of damaged dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry

We report a flow cytometric fluorescence lifetime-based method to discrimin ate damaged/dead from viable cells in immunofluorescently labeled populatio ns using propidium iodide as a dye-exclusion viability probe. Fluorescence signals from propidium iodide and the anti-thymus cell-surface immunofluore scence marker fluorochromes, phycoerythrin and phycoerythrin/Texas Red (tan dem conjugate), which have overlapping emission spectra with propidium iodi de, are resolved based on differences in their fluorescence emission lifeti mes using phase-sensitive detection. Mouse thymus cell samples were first l abeled separately with anti-Thy 1.2 antibody directly conjugated to phycoer ythrin and to phycoerythrin/Texas Red and propidium iodide. Labeled cells w ere then analyzed to determine the lifetimes of the immunofluorescence mark ers and propidium iodide. Based on these results, rat and mouse thymocytes labeled with anti-Thy 1.1 conjugated to phycoerythrin and anti-Thy 1.2 conj ugated to phycoerythrin/Texas Red, respectively, were suspended in phosphat e buffered saline containing propidium iodide, and were analyzed as they pa ssed through a flow chamber and crossed a high-frequency, intensity-modulat ed (sinusoidal) laser excitation beam. The resulting immunofluorescence and propidium iodide signals were resolved based on differences in fluorescenc e lifetimes expressed as phase shifts using phase-sensitive detection and d isplayed as frequency distribution histograms and bivariate contour diagram s. This technology provides a new method to resolve immunofluorescence and propidium iodide signals from overlapping fluorescence emission spectra and a flow cytometric lifetime-based technique to quantify damaged/dead cells in immunofluorescence studies. (C) 1999 Elsevier Science B.V. All rights re served.